Enhancing Photosynthesis

ABSTRACT

Certain embodiments of the present invention relate to methods and products for enhancing the rate of photosynthesis in a plant. In addition, certain embodiments relate to transgenic plants and progeny thereof which comprise an exogenous Rieske Iron Sulphur protein.

FIELD OF THE INVENTION

Certain embodiments of the present invention relate to methods andproducts for enhancing the rate of photosynthesis in a plant. Inaddition, certain embodiments relate to transgenic plants and progenythereof which comprise an exogenous Rieske Iron Sulphur protein.

BACKGROUND TO THE INVENTION

Increasing food and fuel demands caused by a growing world populationhas led to the need to develop higher yielding crop varieties. It hasbeen estimated that by 2050, a 50% increase in the yield of grain cropssuch as wheat and rice will be required if food supply is to meetincreasing demands (Fischer and Edmeades, 2010). A determinant of cropyield is the cumulative rate of photosynthesis over the growing seasonwhich is the result of the crop's ability to capture light, theefficiency by which this light is converted to biomass and how muchbiomass is converted into the usable product e.g. grain in the case ofwheat and rice or biomass in the case of Tobacco. However, the maximumpotential yield of a crop over the growing season under optimalconditions is influenced by both genetic factors and agronomic practice.

A number of strategies have been proposed for increasing the yield ofcrops including selective breeding of plants with traits such as kernelnumber, stomatal conductance, maximum photosynthesis rate, and carbonisotope discrimination in mind. Increasing photosynthetic capacity hasbeen discussed in the art, although it is considered that it is alreadya well-optimised process and that increasing leaf photosynthesis ratewill not necessarily lead to enhanced crop yield (Richards, (2000)Experimental Botany, 51, 447-458; Zhao et al 2008, Plant Science 174,618-625). In addition, this field has been held back due to the lack ofcorrelation between leaf photosynthesis and yield, coupled with evidencethat yield is sink rather than source limited have led to a pervasiveview that crop yields cannot be improved by increasing leafphotosynthetic rates (Long et al 2006).

There are numerous potential targets for increasing yield by way ofenhancing photosynthetic capacity. For example, C3 plants fixatmospheric CO₂ using the Calvin-Benson cycle enzymeribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). One approachincludes improving photosynthetic carbon fixation by increasing theactivity of Calvin-Benson cycle enzymes (Lefebvre et al., 2005; Raines,2011; Simkin et al., 2015). One method is to target Rubisco e.g. togenetically engineer a more efficient Rubisco, or to increase thethermotolerance of Rubisco activase.

There are a number of other parallel systems that have yet to beexplored which may impact on the efficiency of the two photosystems, PSIand PSII. For example, the cytochrome b6f (cyt b6f) complex ofphotosynthetic electron transport acts in both linear electron transport(between PSI and PSII for the generation of ATP and NADPH) and cyclicelectron transport (generation of a trans-membrane proton gradient forATP generation) thereby providing ATP and NADPH for photosyntheticcarbon fixation. The electron flow through the Cyt b6/f complex isconsidered to be a key rate-limiting step for RuBP regeneration (Yamoriet al., 2011).

The cytochrome b6f complex is a 220 kDa symmetric dimer, with eachmonomer being composed of eight subunits. Six of the subunits, PetA(cytochrome f: AtCG00540), PetB (cytochrome b6: AtCG00720), PetD(AtCG00730), PetG (AtCG00600), PetL (AtCG00590) and PetN (AtCG00210) areencoded in the chloroplast genome and 2, PetC (RieskeFeS At4G03280) andPetM (At2G26500), are encoded in the nuclear genome (Cramer et al.,2006; Baniulis et al., 2009; Schottler et al., 2015).

Previous studies have indicated that there is correlation between thecapacity of electron transport and the content of the cyt b6f complex.Initially, cyt b6f inhibitors (Kirchhoff et al., 2000) and latertransgenic anti-sense studies suppressing the accumulation of theRieskeFeS protein (PetC), a key component of the cyt b6f complex (Priceet al., 1995, 1998; Anderson et al., 1997; Yamori et al., 2011), bothdemonstrated a proportional relationship between linear electron fluxand carbon assimilation in leaves, establishing a flux controlcoefficient close to one for the cyt b6f complex (Price et al., 1995;Kirchhoff et al., 2000). In addition, RNAi repression of PetM, one ofthe subunits of cytochrome b6f, resulted in a similar phenotype to thatshown with RNAi repression of Rieske iron sulphur protein (Hojka et al,Plant Physiology, August 2014, Vol. 165, pp. 1632-1646.)

However, there has been no indication that overexpression of the Rieskeiron sulphur protein can lead to increase photosynthesis rate and/orenhanced yield.

This may be due, at least in part, to the recognised complexity of thephotosynthetic pathways and particularly the complexity of thecytochrome b6f complex itself. For example, the cytochrome b6f complexcomprises both nuclear and chloroplast encoded subunits. Furthermore, itis believed that there is strong posttranscriptional control of complexsynthesis and assembly in the chloroplast.

It is an aim of certain embodiments of the present invention to at leastpartially mitigate the problems associated with the prior art.

It is an aim of certain embodiments of the present invention to providea method for improving yield of a plant.

It is an aim of certain embodiments of the present invention to providea method and/or products for upregulating a plurality of components in acytochrome b6f complex in a plant.

SUMMARY OF CERTAIN EMBODIMENTS OF THE INVENTION

In a first aspect of the present invention, there is provided a methodof increasing a photosynthesis rate of a plant, the method comprisingexpressing within said plant an exogenous Rieske iron sulphur protein toform a transgenic plant.

The cytochrome b6f complex and chloroplast ATP synthase form thepredominant sites of photosynthetic flux control. It is considered thatthe inventors have determined for the first time that overexpression ofthe Rieske iron sulphur protein may be sufficient to achieve the goal ofincreasing photosynthesis and yield. Previously, it has been consideredthat overexpression of a single subunit in the cytb6f complex would beinsufficient to achieve this goal. In addition, evidence also suggestedthat the co-regulation of ATP synthase and the cytochrome b6f complexwould be essential to optimise the functions of the thylakoid membrane.Furthermore, it is not known which steps of cytochrome b6f complexbio-genesis ultimately control formation of the complex and there isalso a belief that it is most likely the level of cytochrome b6 thatcoordinates synthesis.

In a second aspect of the present invention, there is provided a methodof producing a transgenic plant having an increased photosynthesis rate,the method comprising expressing within said plant an exogenous Rieskeiron sulphur protein.

In certain embodiments, the transgenic plant has an increasedphotosynthesis rate as compared to a control plant.

In certain embodiments, the transgenic plant has a greater size, greaterbiomass and/or faster growth rate as compared to a control plant. Incertain embodiments, the control plant is a wild-type plant of the samevariety as the transgenic plant.

In certain embodiments, the method further comprises increasing a levelof expression of an endogenous cytochrome b6f complex protein.

In certain embodiments, the method further comprises increasing a levelof expression of one or more of an endogenous chlorophyll a-b bindingprotein (Lhca1), endogenous photosystem I subunit PsaA protein,endogenous photosystem II subunit PsbD protein, endogenous photosystemII subunit PsbA protein and/or endogenous ATP synthase subunit betaprotein (AtpD).

In certain embodiments, the method further comprises the step ofcultivating the transgenic plant under conditions suitable forphotosynthesis to occur.

In certain embodiments, the method further comprises the step ofdetermining an increased photosynthesis rate of the transgenic plant.

In certain embodiments, the method comprises a step of nucleartransformation of a plant cell with a nucleic acid molecule encoding aRieske iron sulphur protein.

In a third aspect of the present invention, there is provided a methodof enhancing yield of a plant, the method comprising expressing withinsaid plant an exogenous Rieske iron sulphur protein to form a transgenicplant.

In certain embodiments, the transgenic plant has an enhanced yield ascompared to a control plant. In certain embodiments, the control plantis a wild-type plant of the same variety as the transgenic plant.

In certain embodiments, the enhanced yield comprises a greater size, agreater biomass and/or faster growth rate as compared to the controlplant.

In certain embodiments, the method comprises a step of nucleartransformation of a plant cell with a nucleic acid molecule encoding aRieske iron sulphur protein.

In certain embodiments, the method further comprises:

-   -   introducing into at least one plant cell an expression vector        comprising a nucleic acid molecule encoding the exogenous Rieske        iron sulphur protein; and optionally    -   regenerating a transgenic plant from said at least one plant        cell.

In certain embodiments, the expression vector is a plasmid suitable fornuclear transformation of the nucleic acid encoding the Rieske ironsulphur protein. In certain embodiments, the expression vector is not aplasmid which directs transformation of the nucleic acid to the plastid.

In certain embodiments, the method comprises nuclear transformation ofthe at least one plant cell with a nucleic acid molecule encoding theexogenous Rieske iron sulphur protein.

In certain embodiments, the method further comprises increasing a levelof expression of an endogenous cytochrome b6f complex protein.

In certain embodiments, the method further comprises the step ofcultivating the transgenic plant under conditions suitable forphotosynthesis to occur.

In certain embodiments, the method further comprises the step ofdetermining an increased photosynthesis rate of the transgenic plant.

In certain embodiments, the method further comprises:

-   -   cultivating the transgenic plant under conditions promoting        plant growth and development; and    -   optionally obtaining a progeny of said transgenic plant.

In certain embodiments, the method further comprises the step ofcultivating the progeny of the transgenic plant under conditionssuitable for photosynthesis to occur.

In certain embodiments, the method further comprises the step ofdetermining an increased photosynthesis rate of the progeny of thetransgenic plant.

In certain embodiments, the step of obtaining a progeny of saidtransgenic plant comprises selfing the transgenic plant therebyproducing a plurality of first generation progeny plants.

In certain embodiments, the method further comprises the step ofcultivating the plurality of first generation progeny plants underconditions suitable for photosynthesis to occur.

In certain embodiments, the method further comprises the step ofdetermining an increased photosynthesis rate of the plurality of firstgeneration progeny plants.

In a further aspect of the present invention, there is provided a methodof increasing expression of a cytochrome b6f complex protein in at leastone plant cell, the method comprising:

-   -   introducing into the at least one plant cell a nucleic acid        molecule encoding an exogenous Rieske iron sulphur protein; and    -   expressing said Rieske iron sulphur protein in said plant cell.

In certain embodiments, the method comprises a step of nucleartransformation of the at least one plant cell with a nucleic acidmolecule encoding a Rieske iron sulphur protein.

In certain embodiments, the cytochrome b6f complex protein is selectedfrom PetA, PetB and a combination of PetA and PetB.

In certain embodiments, the cytochrome b6f complex protein is anendogenous cytochrome b6f complex protein.

In certain embodiments, the step of introducing into at least one plantcell a nucleic acid molecule encoding an exogenous Rieske iron sulphurprotein comprises introducing an expression vector comprising thenucleic acid molecule encoding the exogenous Rieske iron sulphurprotein.

In certain embodiments, the method further comprises the step ofcultivating the transgenic plant under conditions suitable forphotosynthesis to occur.

In certain embodiments, the method further comprises the step ofdetermining an increased photosynthesis rate of the transgenic plant.

In certain embodiments, the method further comprises regenerating atransgenic plant from said at least one plant cell comprising saidnucleic acid molecule.

In certain embodiments, the method further comprises selecting atransgenic plant having an increased expression of a cytochrome b6fcomplex protein relative to a corresponding control plant.

In certain embodiments, the method further comprises selecting atransgenic plant having an increased photosynthesis rate relative to acorresponding control plant.

In certain embodiments, the control plant is a wild-type plant of thesame variety as the transgenic plant.

Aptly, the exogenous Rieske iron sulphur protein comprises an amino acidsequence selected from:

-   -   a) an amino acid sequence as set forth in SEQ. ID. No. 1.

Aptly, the exogenous Rieske iron sulphur protein is a protein having anamino acid sequence which has at least 70% sequence identity to theamino acid set forth in SEQ. ID. No. 1.

In certain embodiments, the method further comprises introducing intothe at least one cell a nucleic acid molecule comprising a nucleic acidsequence selected from:

-   -   a) a nucleic acid sequence as set forth in SEQ. ID. No. 2 or        SEQ. ID. No. 3;    -   b) a nucleic acid sequence having at least 70% sequence identity        to the nucleic acid sequence as set forth in SEQ. ID. No. 2 or        SEQ. ID. No. 3 and which encodes a Rieske iron sulphur protein;    -   c) a nucleic acid sequence which hybridises to (a) or (b) and        which encodes a Rieske iron sulphur protein; and    -   d) a nucleic acid sequence which differs from (a), (b) or (c) by        virtue of the degeneracy of the genetic code and which encodes a        Rieske iron sulphur protein.

In certain embodiments, the nucleic acid molecule is an isolated nucleicacid molecule.

Aptly, the expression vector is a plasmid suitable for nucleartransformation of the nucleic acid encoding the Rieske protein. Incertain embodiments, the expression vector is not a plasmid whichdirects transformation of the nucleic acid to a plastid of the at leastone cell.

In a yet further aspect of the present invention, there is provided achimeric construct for increased expression of a cytochrome b6f proteinin a plant, the chimeric construct comprising a nucleic acid molecule,which encodes a Rieske iron sulphur protein, operably linked to apromoter which is operable in a plant cell, wherein the nucleic acidmolecule comprises a nucleic acid sequence selected from:

-   -   i) a nucleic acid sequence as set forth in SEQ. ID. No. 2 or        SEQ. ID. No. 3;    -   ii) a nucleic acid sequence having at least 70% sequence        identity to the nucleic acid sequence as set forth in SEQ. ID.        No. 2 or SEQ. ID. No. 3 and which encodes a Rieske iron sulphur        protein;    -   iii) a nucleic acid sequence which hybridises to (a) or (b) and        which encodes a Rieske iron sulphur protein; and    -   iv) a nucleic acid sequence which differs from (a), (b) or (c)        by virtue of the degeneracy of the genetic code and which        encodes a Rieske iron sulphur protein.

In certain embodiments, the chimeric construct is a plasmid suitable fordirecting nuclear transformation of the nucleic acid encoding the Rieskeiron sulphur protein. In certain embodiments, the chimeric construct isnot a plasmid which directs transformation of the nucleic acid to theplastid.

In a yet further aspect of the present invention, there is provided aplant cell comprising a nucleic acid molecule comprising a nucleic acidsequence selected from:

-   -   a) a nucleic acid sequence as set forth in SEQ. ID. No. 2 or        SEQ. ID. No. 3;    -   b) a nucleic acid sequence having at least 70% sequence identity        to the nucleic acid sequence as set forth in SEQ. ID. No. 2 and        which encodes a Rieske iron sulphur protein;    -   c) a nucleic acid sequence which hybridises to (a) or (b) and        which encodes a Rieske iron sulphur protein; and    -   d) a nucleic acid sequence which differs from (a), (b) or (c) by        virtue of the degeneracy of the genetic code, wherein the        nucleic acid molecule encodes an exogenous Rieske iron sulphur        protein.

Aptly, the nucleic acid molecule is comprised in an expression vector.Aptly, the expression vector further comprises a promoter.

In certain embodiments, the promoter is a 35s tobacco mosaic viruspromoter.

Aptly, the expression vector is a plasmid. Aptly, the nucleic acidmolecule is expressed in the nucleus.

In a yet further aspect of the present invention, there is provided aplant which is regenerated from the plant cell described herein. Aptly,the plant is a monocotyledonous plant.

Aptly, the plant is selected from wheat, barley, rice and canola.

In a further aspect of the present invention, there is provided use of aplant cell which comprises a nucleic acid molecule comprising a nucleicacid sequence selected from:

-   -   a) a nucleic acid sequence as set forth in SEQ. ID. No. 2 or        SEQ. ID. No. 3;    -   b) a nucleic acid sequence having at least 70% sequence identity        to the nucleic acid sequence as set forth in SEQ. ID. No. 2 and        which encodes a Rieske iron sulphur protein;    -   c) a nucleic acid sequence which hybridises to (a) or (b) and        which encodes a Rieske iron sulphur protein; and    -   d) a nucleic acid sequence which differs from (a), (b) or (c) by        virtue of the degeneracy of the genetic code, in the generation        of a plant that expresses an exogenous Rieske iron sulphur        protein.

In certain embodiments, the plant has one or more of the followingcharacteristics:

-   -   a) enhanced yield;    -   b) increased photosynthesis rate; and/or    -   c) increased expression of a cytochrome bf6 complex protein.

In certain embodiments, the cytochrome bf6 complex protein is anendogenous cytochrome b6f complex protein.

In certain embodiments, the enhanced yield comprises one or more of:

-   -   a) greater biomass;    -   b) greater size; and/or    -   c) greater growth rate, as compared to a control plant.

In certain embodiments, the control plant is a wild-type plant of thesame variety as the plant that expresses the exogenous Rieske ironsulphur protein.

Thus, in certain embodiments of the present invention, there is providedmethods and products which have utility in upregulating one or morecomponents of a cytochrome b6f complex in a plant. The cytochrome b6fcomplex is an eight subunit complex which forms part of thephotosynthetic electron transport chain and acts in both linear electrontransport (between PSI and PSII for the generation of ATP and NADPH) andcyclic electron transport (generation of a trans-membrane protongradient for ATP generation) thereby providing ATP and NADPH forphotosynthetic carbon fixation.

The cytochrome b6f complex is composed of 8 different subunits, 6 ofwhich, PetA (cytochrome f: AtCG00540), PetB (cytochrome b6: AtCG00720),PetD (AtCG00730), PetG (AtCG00600), PetL (AtCG00590) and PetN(AtCG00210) are encoded in the chloroplast genome and 2, PetC (RieskeFeSAt4G03280) and PetM (At2G26500), are encoded in the nuclear genome(Cramer et al., 2006; Baniulis et al., 2009; Schottler et al., 2015).

The complex functions as a dimer with a proposed molecular weight ofapprox. 220 kDA with each monomer composed of the eight subunits. Thetransmembrane domain of the reiskeFeS protein (PetC) and thetransmembrane helices of cytochrome b6 (PetB) are directly implicated inthe monomer-monomer interaction and stability of the complex (Hager etal., 1999; Schwenkert et al., 2007; Hojka et al., 2014), whereas thepetD gene product, is believed to function as a scaffold (Cramer et al.,2006). Furthermore, the PetL gene product is thought to only play a rolein complex stability (Schottler et al, 2007). In contrast, PetG, PetNand PetM have all been described as being essential for both cytochromeb6f assembly and stability. (Bruce and Malkin, 1991; Kuras and Wollman,1994; Hager et al., 1999; Monde et al., 2000; Schwenkert et al., 2007;Hojka et al., 2014).

As used herein, the term “Rieske iron sulphur protein” isinterchangeable with the term “RieskeFeS protein” and “PetC”. Theprotein is encoded by the petC gene. The term refers to a RieskeFeSprotein of any plant species including for example Nicotiana tabacum.Thus, certain embodiments of the present invention involve the use of aRieskeFeS protein provided it is an exogenous protein.

The amino acid sequence of a N. tabacum RieskeFeS protein is shown inFIG. 12 and is referred to herein as SEQ ID. No. 1. The amino acidsequence can also be found under accession number: X64353.1http://www.ncbi.nlm.nih.gov/nuccore/X64353

In certain embodiments, the RieskeFeS protein is a protein having anamino acid sequence as depicted in SEQ. ID. No. 1 or a variant thereof.Variants are discussed herein.

The cDNA sequence of N. tabacum PetC which encodes the RieskeFeS proteinof FIG. 12 is shown in FIG. 13 and referred to herein as SEQ. ID. No. 2.The N. tabacum PetC gene sequence is shown in FIG. 14 and referred toherein as SEQ. ID. No. 3. Certain embodiments of the present inventioncomprise the use of a nucleic acid molecule comprising the nucleic acidsequence of SEQ. ID. No. 2 or No. 3 or variants thereof. Variants aredescribed herein.

Certain embodiments of the present invention comprise the use of aRieskeFeS protein from a plant species other than N. tabacum. Forexample, in certain embodiments, the RieskeFeS protein is fromArabidopsis thaliana. Aptly, the RieskeFeS protein is from for example aperennial monocotyledonous or dicotyledonous plant, including by way ofexample, Triticum species (wheat), Zea mays (maize), Hordeum, Oryzasativa (rice), Borago officinalis (borage), Camelina (False flax),Brassica species such as B. campestris, B. napus, B. rapa, B, carinata(mustard, oilseed rape or turnip rape; Cannabis sativa (hemp); Carthamustinctorius (safflower); Cocos nucifera (coconut); Crambe abyssinica(crambe); Cuphea species; Elaeis guinensis (African oil palm); Elaeisoleifera (American oil palm); Glycine max (soybean); Helianthus annuus(sunflower); Linum usitatissimum (linseed or flax); Oenothera biennis(evening primrose); Olea europaea (olive). Aptly, the RieskeFeS proteinis exogenous to the plant species into which it is introduced.

Thus, in certain embodiments of the present invention, the RieskeFeSprotein is a homolog to the RieskeFeS protein of FIG. 12. The term“homolog” is a generic term of the art used to indicate a polypeptide orpolynucleotide sequence possessing a high degree of sequence relatednessto a subject sequence. Such relatedness may be quantified by determiningthe degree of identity and/or similarity between the sequences beingcompared.

As used herein, the term “percent sequence identity” is the percentageof amino acids or polynucleotides that are identical when the twosequences are compared. Homology or sequence identity of two amino acidsequence or of two nucleic acid sequences may be determined by methodsknown in the art. For example, the sequence identity may be determinedusing the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad.Sci., U.S.A 87: 2264-2268. Such an algorithm is incorporated into theNBLAST and XBLAST programs of Altschul et al. (1990) J. Mol. Biol. 215:403-410. BLAST nucleotide searches are performed with the NBLASTprogram, score=100, word length 12, to obtain nucleotide sequenceshomologous to a nucleic acid molecules of certain embodiments of thepresent invention. BLAST protein sequences are performed with the XBLASTprogram, score=50, word length=3, to obtain amino acid sequenceshomologous to a reference polypeptide (e.g. SEQ. ID. No 1). To obtaingapped alignments for comparison purposes, Gapped BLAST is utilised asdescribed in Altshul et al. (1997) Nucleic Acids Res. 25: 3389-3402.When utilising BLAST and Gapped BLAST programs, the default parametersare typically used. (See http://www.ncbi.nlm.nih.gov).

In certain embodiments, the RieskeFeS protein comprises an amino acidsequence which is approx. 70% identical or greater to the amino acidsequence of SEQ. ID. No 1. In certain embodiments, the RieskeFeS proteinis at least 80% identical to the amino acid sequence of SEQ. ID. No. 1.In certain embodiments, the RieskeFeS protein is at least 90% identicalto the amino acid sequence of SEQ. ID. No 1, e.g. 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence depicted in SEQ. ID. No. 1.

In certain embodiments, the RieskeFeS protein is encoded by a genecomprising a nucleotide sequence which is at least 85% identical to thenucleotide sequence depicted in SEQ. ID. No 3. For example, in certainembodiments, the RieskeFeS protein is encoded by a gene which comprisesa nucleotide sequence having a sequence identity of at least 70%identical, e.g. 80% identical, e.g. 90% to the nucleotide sequence ofSEQ. ID. No. 3 e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or100% identical to the nucleotide sequence depicted in SEQ. ID. No. 3.

Typically, homologous sequences can be confirmed by hybridization,wherein the hybridization takes place under stringent conditions. Usingthe stringent hybridization (i.e. washing the nucleic acid fragmentstwice where each wash is at room temperature for 30 minutes with 2×sodium chloride and sodium citrate (SCC buffer; 1.150. mM sodiumchloride and 15 mM sodium citrate, pH 7.0) and 0.1% sodium dodecylsulfate (SDS); followed by washing one time at 50° C. for 30 minuteswith 2×SCC and 0.1% SDS; and then washing two times where each wash isat room temperature for 10 minutes with 2×SCC), homologous sequences canbe identified comprising at most about 25 to about 30% base pairmismatches, or about 15 to about 25% base pair mismatches, or about 5 toabout 15% base pair mismatches.

In certain embodiments, the RieskeFeS protein is a naturally occurringvariant of the protein having an amino acid sequence as depicted in SEQ.ID. No. 1. Non-naturally occurring variants that differ from the proteinhaving an amino acid sequence as depicted in SEQ. ID. No. 1 and have thebiological function of a RieskeFeS protein are also encompassed bycertain embodiments of the present invention.

In certain embodiments, the RieskeFeS protein comprises one or moreconservative amino acid substitution changes, i.e. changes of similarlycharged or uncharged amino acids. Genetically encoded amino acids aregenerally divided into four families:

(1) acidic (aspartate, glutamate);

(2) basic (lysine, arginine, histadine);

(3) non-polar (alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan); and

(4) uncharged polar (glycine, asparagine, glutamine, cysteine, serine,threonine, tyrosine).

As each member of a family has a similar physical and chemicalproperties as the other members of the same family, it is reasonable toexpect that an isolated replacement of a leucine with an isoleucine orvaline, a threonine with a serine or a similar replacement of an aminoacid with a structurally related amino acid will not have a major effecton the binding properties of the resulting molecule.

The terms “isolated” or “purified”, used interchangeably herein, refersto a nucleic acid, a polypeptide, or other biological moiety that isremoved from components with which it is naturally associated. The term“isolated” can refer to a polypeptide that is separate and discrete fromthe whole organism with which the molecule is found in nature or ispresent in the substantial absence of other biological macro-moleculesof the same type. The term “isolated” with respect to a polynucleotidecan refer to a nucleic acid molecule devoid, in whole or in part, ofsequences normally associated with it in nature; or a sequence, as itexists in nature but having heterologous sequences in associatedtherewith; or a molecule dissociated from the chromosome. Purity andhomogeneity are typically determined using analytical chemistrytechniques, for example, polyacrylamide gel electrophoresis or highperformance liquid chromatography. In some embodiments, the term“purified” means that the nucleic acid or protein is at least 85% pure,e.g. at least 90% pure, e.g. 95% pure.

In certain embodiments, a gene encoding a RieskeFeS protein may beexpressed in vectors suitable for in vivo expression such as for exampleplant expression systems. Aptly, the vector is a recombinant expressionvector. The term “recombinant expression vector” as used herein refersto a plasmid, virus, cosmid, baculovirus, bacterial, yeast or viralvector or other means known in the art that has been manipulated byinsertion or incorporation of the PetC gene which encodes the RieskeFeSprotein. Recombinant expression vectors e.g. plasmids for use in certainembodiments of the present invention may be commercially available,publically available on an unrestricted basis, or can be constructedfrom available plasmids by routine application of well-known, publishedprocedures. Such vectors can be transformed into suitable host cells toform transgenic plants or parts thereof. Suitable vectors include forexample gateway-cloning-compatible plant destination vectors forexpression of proteins in transgenic plants. Aptly, the vector is aplasmid suitable for nuclear transformation of the gene.

In certain embodiments, the vector is a pGWB2 gateway vector.

The vector is aptly adapted for expression in a plant cell and maycomprise one or more regulatory elements necessary for expression of thenucleic acid molecule in a plant operatively linked to the nucleic acidmolecule encoding a RieskeFeS protein. The term “operatively linked” asused herein refers to a juxtaposition wherein the components sodescribed are in a relationship permitting them to function in theirintended manner.

Certain embodiments of the present invention comprise the use of apromoter and/or other control sequences e.g. enhancers, transcriptionterminators and the like. A promoter may direct transcription of thegene in question. A promoter may be inducible or constitutive.

In certain embodiments of the present invention, the promoter is a 35Stobacco mosaic virus promoter.

In certain embodiments, the vector contains the RieskeFeS codingsequence operably linked to a constitutive promoter (e.g. 35s, orFigwort Mosaic Virus promoter). In other embodiments, the codingsequence is operably linked to an inducible promoter. In otherembodiments, the coding sequence is operably linked to a tissue specificpromoter, which is a photosynthetic tissue specific promoter in someembodiments.

In certain embodiments, the vector may further comprise a selectablemarker, that is, a gene encoding a protein necessary for the survival orgrowth of a host cell transformed with the vector. Such a marker genemay be co-introduced into the host cell or may be containing on thecloning vector. Suitable selection genes encode proteins that conferresistance to antibiotics or other toxic substances for example. Manyselection genes are known and could be used with the present inventionincluding, but not limited to, neo, which provides kanamycin resistanceand can be selected for using kanamycin, G418, paromomycin, etc.; bar,which confers bialaphos or phosphinothricin resistance; a mutant EPSPsynthase protein conferring glyphosate resistance; a nitrilase such asbxn from Klebsiella ozaenae which confers resistance to bromoxynil; amutant acetolactate synthase (ALS) which confers resistance toimidazolinone, sulfonylurea or other ALS inhibiting chemicals; amethotrexate resistant DHFR, a dalapon dehalogenase that confersresistance to the herbicide dalapon; or a mutated anthranilate synthasethat confers resistance to 5-methyl tryptophan.

In certain embodiments, the vector comprises one or more genes whichencode a protein that confers resistance to kanamycin and/or hygromycin.

With respect to a coding sequence, the term “plant-expressible” meansthat the coding sequence (nucleotide sequence) can be efficientlyexpressed by plant cells, tissue and/or whole plants. As used herein, aplant-expressible coding sequence has a GC composition consistent withacceptable gene expression in plant cells, a sufficiently low CpGcomposition so that expression of that coding sequence is not restrictedby plant cells, and codon usage that is consistent with that of plantgenes.

In certain embodiments, the vector may be introduced into a host cell. Atransgenic plant may be regenerated from the host cell. Transgenicplants can be generated using standard plant transformation methodsknown to those skilled in the art. The method of transformation dependsupon the plant to be transformed.

These include, but are not limited to, Agrobacterium vectors,polyethylene glycol treatment of protoplasts, biolistic DNA delivery, UVlaser microbeam, gemini virus vectors or other plant viral vectors,calcium phosphate treatment of protoplasts, electroporation of isolatedprotoplasts, agitation of cell suspensions in solution with microbeadscoated with the transforming DNA, agitation of cell suspension insolution with silicon fibers coated with transforming DNA, direct DNAuptake, liposome-mediated DNA uptake, and the like. Such methods havebeen published in the art. See, e.g., Methods for Plant MolecularBiology (Weissbach & Weissbach, eds., 1988); Methods in Plant MolecularBiology (Schuler & Zielinski, eds., 1989); Plant Molecular BiologyManual (Gelvin, Schilperoort, Verma, eds., 1993); and Methods in PlantMolecular Biology—A Laboratory Manual (Maliga, Klessig, Cashmore,Gruissem & Vamer, eds., 1994).

Agrobacterium vectors are often used to transform dicot species.Agrobacterium binary vectors include, but are not limited to, BIN19 andderivatives thereof, the pBI vector series, and binary vectors pGA482,pGA492, pLH7000 (GenBank Accession AY234330) and any suitable one of thepCAMBIA vectors (derived from the pPZP vectors constructed byHajdukiewicz, Svab & Maliga, (1994) Plant Mol Biol 25: 989-994,available from CAMBIA, GPO Box 3200, Canberra ACT 2601, Australia or viathe worldwide web at CAMBIA.org). For transformation of monocot species,biolistic bombardment with particles coated with transforming DNA andsilicon fibers coated with transforming DNA are often useful for nucleartransformation. Alternatively, Agrobacterium “superbinary” vectors havebeen used successfully for the transformation of rice, maize and variousother monocot species. The vector may be introduced into the host cellusing techniques known in the art, for example floral dipping and/orfloral (Arabidopsis), Agrobacterium infection (tobacco, tomato, soya,casava) or partical bombardment (wheat, barley).

By “transformation”, it is meant a permanent or transient genetic changeinduced in a cell following incorporation of exogenous DNA to the cell.Thus, in certain embodiments, the host cell is transformed withexogenous DNA comprising inter alia a sequence which encodes a RieskeFeSprotein.

In certain embodiments, the vector used for transformation is a plasmid.A plasmid may be expressed in the nucleus of a plant cell rather than ina plastid of a plant cell, such as a chloroplast, as would occur if aplastid vector is used. Thus in certain embodiments the exogenous ReiskeFeS protein is expressed in the nucleus of the plant cell.

As noted above, a transgenic plant may be regenerated from a transformedplant cell. As used herein, a “transgenic plant” is a plant which hasbeen genetically modified to contain and express recombinant DNAsequences, either a regulatory RNA molecules or as proteins. In certainembodiments, the transgenic plant is genetically modified to express aDNA molecule which encodes an exogenous RieskeFeS protein. As usedherein, the term “transgenic plant” also encompasses progeny of aninitial transgenic plant, wherein the progeny contain and are capable ofexpressing the RieskeFeS encoding gene sequence. Additionally, seeds andother plant parts are encompassed within the definition of “transgenicplant” as used herein.

Aptly, the transgenic plant overexpresses the RieskeFeS protein. That isto say, in certain embodiments, the transgenic plant which comprises oneor more cells which have been transformed and/or comprise an exogenousRieskeFeS protein, has a level of expression of RieskeFeS protein whichis greater than a control plant. The control plant is aptly a plant ofthe same species as the transgenic plant and which does not comprise anexogenous RieskeFeS protein. Aptly, the control plant is a wild-typeplant of the same species as the transgenic plant. In certainembodiments, the control plant is a null segregant in whichnon-integration of the transgene has been determined.

The transgenic plant may express RieskeFeS protein at a level which isat least 105% e.g. 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%,155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195%, 200% or more ascompared to a control plant e.g. a wild-type plant.

The inventors have surprisingly discovered that overexpression of theRieskeFeS protein e.g. by way of introduction of an exogenous gene whichencodes a ReiskeFeS protein, expression of other components of theplant's endogenous cytochrome b6f complex is upregulated.

Thus, in certain embodiments, the transgenic plant further comprises alevel of expression of endogenous PetA and/or PetB which is greater thanthe level of expression of PetA and/or PetB of a wild type plant whichdoes not comprise an exogenous RieskeFeS protein. Thus, in certainembodiments, the transgenic plant comprises a level of PetA expressionwhich is at least 105% or greater as compared to the level of PetAexpression in a control plant e.g. a wild-type plant. In certainembodiments, the transgenic plant expresses PetA at a level which is atleast 105% e.g. 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%,155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195%, 200% or more ascompared to a control plant e.g. a wild-type plant.

In certain embodiments, the transgenic plant comprises a level of PetBexpression which is at least 105% or greater as compared to the level ofPetB expression in a control plant e.g. a wild-type plant. Aptly, thetransgenic plant expresses PetB at a level which is at least 105% e.g.110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%,170%, 175%, 180%, 185%, 190%, 195%, 200% or more as compared to acontrol plant e.g. a wild-type plant.

The amino acid sequence of PetA and PetB proteins will vary according tothe species of transgenic plant. By way of example, PetA (cytochrome f:AtCG00540), PetB (cytochrome b6: AtCG00720), gene and protein sequencescan be found in PetA http://www.ncbi.nlm.nih.gov/protein/6685374 PetBhttp://www.ncbi.nlm.nih.gov/protein/6685349

It was also discovered that other proteins involved in electrontransport such as chlorophyll a-b binding protein (Lhca1), endogenousphotosystem I subunit PsaA protein, endogenous photosystem II subunitPsbD protein, endogenous photosystem II subunit PsbA protein andendogenous ATP synthase subunit beta protein (AtpD) may also beupregulated.

Thus, in certain embodiments, the transgenic plant further comprises alevel of expression of an endogenous protein selected from chlorophylla-b binding protein (Lhca1), endogenous photosystem I subunit PsaAprotein, endogenous photosystem II subunit PsbD protein, endogenousphotosystem II subunit PsbA protein and endogenous ATP synthase subunitbeta protein (AtpD) which is greater than the level of expression ofsuch a protein of a wild type plant which does not comprise an exogenousRieskeFeS protein. Thus, in certain embodiments, the transgenic plantcomprises a level of one or more of chlorophyll a-b binding protein(Lhca1), endogenous photosystem I subunit PsaA protein, endogenousphotosystem II subunit PsbD protein, endogenous photosystem II subunitPsbA protein and endogenous ATP synthase subunit beta protein (AtpD)which is at least 105% or greater as compared to the level of expressionin a control plant e.g. a wild-type plant. In certain embodiments, thetransgenic plant expresses one or more of such proteins at a level whichis at least 105% e.g. 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%,150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195%, 200% or moreas compared to a control plant e.g. a wild-type plant.

As used herein, the term “plant” refers to all genera and species ofhigher and lower plants of the Plant Kingdom. The term includes themature plants, seeds, shoots ad seedlings, and part, propagationmaterial, plant organ tissue, protoplasts, callus and other cultures,for example cell cultures, derived from them, and all other species ofgroups of plant cells giving functional or structural units. Matureplants refers to plants at any developmental stage beyond the seedling.Seedling refers to a young, immature plant at an early developmentalstage.

Plants of any species are included in embodiments of the invention;these include, but are not limited to, tobacco, Arabidopsis and other“laboratory-friendly” species, cereal crops such as maize, wheat, rice,soybean barley, rye, oats, sorghum, alfalfa, clover and the like,oil-producing plants such as canola, safflower, sunflower, peanut, cacaoand the like, vegetable crops such as tomato tomatillo, potato, pepper,eggplant, sugar beet, carrot, cucumber, lettuce, pea and the like,horticultural plants such as aster, begonia, chrysanthemum, delphinium,petunia, zinnia, lawn and turfgrasses and the like.

Certain embodiments of the present invention relate to plants such asfor example perennial monocotyledonous or dicotyledonous plants,including by way of example, Triticum species (wheat), Zea mays (maize),Hordeum, Oryza sativa (rice), Borago officinalis (borage), Camelina(False flax), Brassica species such as B. campestris, B. napus, B. rapa,B, carinata (mustard, oilseed rape or turnip rape; Cannabis sativa(hemp); Carthamus tinctorius (safflower); Cocos nucifera (coconut);Crambe abyssinica (crambe); Cuphea species; Elaeis guinensis (Africanoil palm); Elaeis oleifera (American oil palm); Glycine max (soybean);Helianthus annuus (sunflower); Linum usitatissimum (linseed or flax);Oenothera biennis (evening primrose); Olea europaea (olive).

In certain embodiments, a transgenic plant as described herein will havea greater biomass than a control plant of the same species which iscultivated in similar or identical conditions. In certain embodiments,the transgenic plant comprises a biomass which is at least 20% greaterthan the biomass of a wild-type plant e.g. 30%, 40%, 50%, 60%, 70%, 80%,90%, 100%, 110% or greater.

In certain embodiments, the transgenic plant as described herein shows acharacteristic of yield which is improved as compared to a controlplant. The characteristics may be selected from:

-   -   a) greater biomass;    -   b) greater size; and/or    -   c) greater growth rate, as compared to a control plant.

Therefore, in certain embodiments of the invention it is shown that themanipulation of the cytochrome b6f complex by way of overexpression ofan exogenous RieskeFeS protein has the potential to influencephotosynthesis, growth and seed yield.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

Certain embodiments of the present invention will now be describedhereinafter, by way of example only, with reference to the followingfigures:

FIGS. 1A and B is an image of Western blot gels:

Western blot analysis in fully expanded leaves from three expressinglines. Protein extracts from leaf discs taken from two independentleaves per plant. PetA and PetB are subunits of the cytochrome b6fcomplex. Lhca amd PsaA are PSI proteins and PsbD/D2 and PsbA/D1 are PSIIproteins. AtpD is ATP synthase protein. Controls for protein loadingincluding the calvin cycle enzyme FBP aldolase (FBPA), thephotorespiration protein glycine decarboxylase H-subunit (GDC-H) andRubisco small subunit (Rubisco). B) Wild type proteins were loaded in arange from 0.63 μg to 10 μg and then compared to protein fractionsloaded from lines 9, 10 and 11.

FIG. 1C) The level of each protein in each plant line was statisticallyanalysed against wild type grown plants and is shown as a relativepercentage of wild type levels.

FIG. 2: Determination of photosynthetic capacity and leaf area intransgenic seedlings using fluorescence imaging. WT and transgenicplants were grown in controlled environment conditions with a lightintensity of 130 m⁻² s⁻¹, 8 h light/16 h dark cycle for 14 days andfluorescence imaging used to determine F_(q)′/F_(m)′ (maximum PSIIoperating efficiency) at two light intensities. FluorescenceF_(q)′/F_(m)′ images taken at (a) 310 μmol m⁻² s⁻¹ and (b) 450 μmol m⁻²s⁻¹. (c) A significant decrease in non-photochemical quenching (NPQ) wasalso observed in these plants (d) leaf area at time of analysis and e)is a fluorescence image of the leaves of WT and transgenic plants.

The data was obtained using 4-6 individual plants from each linecompared to 6 WT. Significant differences (p<0.05) are represented ascapital letters indicating if each specific line is significantlydifferent from another. Lower case italic lettering indicates lines arejust below significance (>0.05-<0.1).

FIG. 3: Determination of photosynthetic capacity in transgenic plants at21% O₂. WT and transgenic plants were grown in controlled environmentconditions with a light intensity 130 μmol m⁻² s⁻¹, 8 h light/16 h darkcycle for four weeks and Light Response curves were carried out at 2%O₂. (a) is a light Response Curves for each individual rieskeFeS and (b)is a light response curve for all lines combined compared to wild type(WT). (c) is a graph showing average F_(q)′/F_(m)′ data, (d) is a graphshowing Amax, (e) is a graph showing average stomatal conductance overthe evaluating period. The data was obtained using 4 individual plantsper line compared to 4 WT. (ALL: combined data set of allover-expressing lines). Significant differences (p<0.05) are representedas capital letters indicating if each specific line is significantlydifferent from another.

FIG. 4: Determination of photosynthetic capacity and rate of electrontransport (rETR) in transgenic plants at 2% O₂. WT and transgenic plantswere grown in controlled environment conditions with a light intensity130 μmol m⁻² s⁻¹, 8 h light/16 h dark cycle for four weeks and LightResponse curves were carried out at 2% O₂. A) is a graph showing lightResponse Curves for each individual RieskeFeS ox, B) is a graph showingrETR for individual plant lines compared to wild type and C) is a graphshowing light response curves for all plant lines combined compared towild type (WT). D) is a graph showing average F_(q)′/F_(m)′ data, E) isa graph showing Amax F) is a graph showing average stomatal conductanceover the evaluating period. The data was obtained using 4 individualplants per line compared to 4 WT. (ALL: combined data set of allover-expressing lines). Significant differences (p<0.05) are representedas capital letters indicating if each specific line is significantlydifferent from another.

FIG. 5: Determination of the efficiency of electron transport in theleaves of young Rieske Fes ox plants (27 days after planting). WT andRieske FeS ox plants were grown in controlled environment conditionswith a light intensity of 130 mmol m⁻² s⁻¹, 8 h light/16 h dark cycleand the redox state was determined using a Dual-PAM at a light intensityof 220 mmol m⁻² s⁻¹. The data was obtained using four individual plantsfrom Rieske FeS ox line 11 and compared to WT (five plants). Significantdifferences are indicated (*p<0.05). Bars represent standard errors. Thequantum efficiency of PSI (FIG. 5A), quantum efficiency of PSII (FIG.5B), level of Q_(A) reduction (1-qp) (5C), fraction of open PSII centres(FIG. 5D), NPQ (FIG. 5E) and reduction in stress induced limitation ofNPQ (q_(N)) (FIG. 5F) are all shown.

FIG. 6: Photosynthetic responses of WT and transgenic plants grown ingreenhouse under square wave isolights. Photosynthetic carbon fixationrates were determined as a function of increasing CO₂ concentrations(A/Ci) at saturating-light levels in developing leaves. WT andtransgenic plants were grown in controlled environment conditions with alight intensity 280 μmol m⁻² s⁻¹, 12 h light/12 h dark cycle for fourweeks. (a) is a graph showing A/Ci Curves for each individual rieskeFeSand (b) is a graph showing all lines combined compared to wild type(WT). (c) A_(max)/A_(sat), (d) is a graph showing Rubisco activity and(e) is a graph showing J_(max) derived from A/Ci response curves usingthe equations published by von Caemmerer and Farquhar (1981).Significant differences (<0.05) are represented by capital letters.Lower case italic lettering indicates lines are just below significance(>0.05-<0.1).

FIG. 7: Final leaf area and biomass of WT and transgenic plants weregrown in controlled environment conditions with a light intensity 280μmol m⁻² s⁻¹, 12 h light/12 h dark cycle for four weeks. (a) Total leafarea (b) dry weight (d) area of leaves of all lines. FIG. 7C is aphotograph of the WT and transgenic leaves. Significant differences(p<0.05) are represented by capital letters within the histogram.

FIG. 8: Growth analysis of wild type (WT) and transgenic lines grown inlow light. Plants were grown at 130 μmol m⁻² s⁻¹ light intensity in longdays (8 h/16 h days). (a) is a photograph showing the appearance ofplants at 10 and 18 days after planting. (b) is a graph illustratingplant growth rate evaluated over the first 20 days. (c) is a graphillustrating final biomass at 25 days post planting. % increase overwild type are indicated as numbers within the histogram. Results arerepresentative of 6 to 9 plants from each line. Significantdifferences * (p<0.01), ** (p<0.001), are indicated.

FIG. 9: Schematic representation of the RieskeFeS over-expression vectorpGWRi. RB, T-DNA right border; Pnos, nopaline synthase promoter; NTP II,neomycin phosphotransferase gene; Tnos, nopaline synthase terminator;P35S, rbcS2B promoter (1150 bp; At5g38420). Constructs were used totransform wild Arabidopsis (Col-0).

FIG. 10: Molecular and Biochemical Analysis of the transgenic plantsover-expressing the RieskeFeS protein from tobacco. Western blotanalysis in fully expanded leaves from 27 primary lines. Protein levelswere compared to PCR negative lines (AZ). Lines 9, 10 and 11 wereselected for study and line 19 was used as a negative control in somestudies. FIG. 10B is a photograph of the plant leaves.

FIG. 11: Determination of photosynthetic capacity and leaf area intransgenic seedlings using fluorescence imaging. WT and transgenicplants were grown in controlled environment conditions with a lightintensity of 110 m⁻² s⁻¹, 8 h light/16 h dark cycle for 14 days andfluorescence imaging used to determine F_(q)′/F_(m)′ (maximum PSIIoperating efficiency) at a light intensity of (a) 600 μmol m⁻² s⁻¹. (b)Shows Fv/Fm of dark adapted plants. (The data was obtained using 4-6individual plants from each line compared to 6 WT). Significantdifferences (p<0.05) are represented as capital letters indicating ifeach specific line is significantly different from another.

FIG. 12: illustrates the amino acid sequence of N. tabacum Rieske ironsulphur protein (SEQ. ID. No. 1).

FIG. 13: illustrates the cDNA sequence of N. tabacum petC gene (SEQ. ID.No. 2); and

FIG. 14: illustrate the DNA sequence of N. tabacum petC gene (SEQ. ID.No. 3).

FIG. 15: shows the maximum PSII efficiency (F_(q)′/F_(m)′). (a)F_(q)′/F_(m)′ for each individual rieskeFeS and (b) all lines combinedcompared to wild type (WT).

FIG. 16: non-photochemical quenching (NPQ). (a) NPQ for each individualrieskeFeS and (b) all lines combined compared to wild type (WT).

FIG. 17: maximum potential operating efficiency of PSII in the light(F_(v)′/F_(m)′). (a) F_(v)′/F_(m)′ for each individual rieskeFeS and (b)all lines combined compared to wild type (WT).

FIG. 18: Comparative analysis of Wild type and null segragants used inthis study. WT—wild type plants grown from seed batch. AZY—nullsegragants recovered from segragating lines verified by PCR fornon-integration of the transgene. (a) F_(q)′/F_(m)′ at 200, (b)F_(q)′/F_(m)′ at 600, (c) leaf area at the time of analysis.

EXAMPLES

Materials and Methods

Rieske Iron Sulphur Protein of the Cytochrome b6f (Cyt b6f)

The full-length coding sequence of the Rieske iron sulphur protein ofthe cytochrome b6f (Cyt b6f: X64353) was amplified by RT-PCR usingprimers NtRieskeFeSF (5′caccATGGCTTCTTCTACTCTTTCTCCAG'3 (SEQ ID. No. 4)and NtRieskeFeSR (5′CTAAGCCCACCATGGATCTTCACC'3 (SEQ ID. No. 5). Theresulting amplified product was cloned into pENTR/D (Invitrogen, UK) tomake pENTR-NtRieskeFeS and the sequence was verified and found to beidentical. The full-length cDNA was introduced into the pGWB2 gatewayvector (Nakagawa et al., 2007: AB289765) by recombination from thepENTR/D vector to make pGW-NtRieske (B2-NtRi). cDNA are undertranscriptional control of the 35s tobacco mosaic virus promoter, whichdirects constitutive high-level transcription of the transgene, andfollowed by the nos 3′ terminator. Construct maps are shown in FIG. 9.

Generation of Transgenic Plants

The recombinant plasmid B2-NtRi was introduced into wild typeArabidopsis by floral dipping (Clough and Bent, 1998) usingAgrobacterium tumefaciens GV3101. Positive transformants wereregenerated on MS medium containing kanamycin (50 mg L⁻¹), hygromycin(20 mg L⁻¹). Kanamycin/hygromycin resistant primary transformants (T1generation) with established root systems were transferred to soil andallowed to self fertilize.

Plant Growth Conditions

Wild-type T2 Arabidopsis plants resulting from self-fertilization oftransgenic plants were germinated in sterile agar medium containingMurashige and Skoog salts (plus kanamycin 100 mg for the transformants)and grown to seed in soil (Levington F2, Fisons, Ipswich, UK) and linesof interest were identified by western blot and qPCR. For experimentalstudy, T3 progeny seeds from selected lines were germinated on soil incontrolled environment chambers at an irradiance of 130 μmol photons m⁻²s⁻¹, 22° C., relative humidity of 60%, in an 8 h/16 h square-wavephotoperiod. Plants were sown randomly and trays rotated daily under thelight. Leaf areas were calculated using standard photography and ImageJsoftware (imagej.nih.gov/ij).

Wild type plants used in this study were a combined group of WT and nullsegregants from the transgenic lines verified by PCR for non-integrationof the transgene. No significant differences in growth parameters wereseen between these groups (see FIG. 18).

Protein Extraction and Western Blotting

Four leaf discs (0.6-cm diameter) from two individual leaves, forwestern blot, were taken and immediately plunged into liquid N², andstored at −80° C. Samples were ground in liquid nitrogen and proteinquantification determined (Harrison et al., 1998). Samples were loadedon an equal protein basis, separated using 12% (w/v) SDS-PAGE,transferred to polyvinylidene difluoride membrane, and probed usingantibodies raised against the cytochrome b6 complex proteins PetA(AS08306), PetB (AS03034), and PetC (RieskeFeS: AS08330), PsbA(AS01016), PsaA (AS06172), Lhca1 (AS01005) and against the Glycinedecarboxylase H-subunit (AS05074), all purchased from NewmarketScientific (UK). FBPA antibodies were raised against a peptide from aconserved region of the protein [C]-ASIGLENTEANRQAYR-amide, CambridgeResearch Biochemicals, Cleveland, UK (Simkin et al. 2015). Proteins weredetected using horseradish peroxidase conjugated to the secondaryantibody and ECL chemiluminescence detection reagent (Amersham,Buckinghamshire, UK). Proteins were quantified using a Fusion FX VilberLourmat Imager (Peqlab, Lutterworth, UK).

Chlorophyll Fluorescence Imaging

Chlorophyll fluorescence measurements were performed on 10-day-oldArabidopsis seedlings that had been grown in a controlled environmentchamber providing 130 μmol mol⁻²s⁻¹ photosynthetic photon flux density(PPFD) and ambient CO₂ at 22° C. Chlorophyll fluorescence parameterswere obtained using a chlorophyll fluorescence (CF) imaging system(Technologica, Colchester, UK; Barbagallo et al., 2003; Baker andRosenqvist, 2004). The operating efficiency of photosystem two (PSII)photochemistry, F_(q)′/F_(m)′, was calculated from measurements ofsteady state fluorescence in the light (F′) and maximum fluorescence inthe light (F_(m)′) was obtained after a saturating 800 ms pulse of 6200μmol m⁻² s⁻¹ PPFD using the following equationF_(q)′/F_(m)′=(F_(m)′−F′)/F_(m)′. Images of F_(q)/F_(m)′ were takenunder stable PPFD of 310, 450 and 600 μmol m⁻² s⁻¹ PPFD (Baker et al.,2001; Oxborough and Baker, 1997).

A/C_(i) Response Curves

The response of net photosynthesis (A) to intracellular CO₂ (C_(i)) wasmeasured using a portable gas exchange system (CIRAS-1, PP Systems Ltd,Ayrshire, UK). Leaves were illuminated using a red-blue light sourceattached to the gas-exchange system, and light levels were maintained atsaturating photosynthetic photon flux density (PPFD) of 1000 μmol m⁻²s⁻¹ with an integral LED light source (PP Systems Ltd, Ayrshire, UK) forthe duration of the A/C_(i) response curve. Measurements of A were madeat ambient CO₂ concentration (C_(a)) of 400 μmol mol⁻¹, before C_(a) wasdecreased in a stepwise manner to 300, 200, 150, 100, 50 μmol mol⁻¹before returning to the initial value and increased to 500, 600, 700,800, 900, 1000, 1100, 1200 μmol mol⁻¹. Leaf temperature and vapourpressure deficit (VPD) were maintained at 22° C. and 1±0.2 kParespectively. The maximum rates of Rubisco—(Vc_(max)) and the maximumrate of electron transport for RuBP regeneration (J_(max)) weredetermined and standardized to a leaf temperature of 25° C. based onequations from Bernacchi et al. (2001), and McMurtrie & Wang (1993)respectively.

Photosynthetic Capacity

Photosynthesis as a function of PPFD (A/Q response curves) was measuredusing a Li-Cor 6400XT portable gas exchange system (Li-Cor, Lincoln,Nebr., USA). Cuvette conditions were maintained at a leaf temperature of22° C., relative humidity of 50-60%, and ambient growth CO₂concentration (400 mmol mol⁻¹ for plants grown in ambient conditions).Leaves were initially stabilized at saturating irradiance 1000 μmol m⁻²s⁻¹, after which A and g_(s) was measured at the following PPFD levels;0, 50, 100, 150, 200, 250, 300, 350, 400, 500, 600, 800, 1000 μmol m⁻²s⁻¹. Measurements were recorded after A reached a new steady state (1-2min) and before stomatal conductance (g_(s)) changed to the new lightlevels. A/Q analyses were performed at 21% and 2% O₂.

Gas Exchange Measurements

The response of net photosynthesis (A) to intracellular CO₂ (C_(i)) wasmeasured using a portable gas exchange system (cirus 1). The gasexchange system was zeroed daily using silica gel to remove water andsoda lime (sofnolime, Morgan Medical, Kent, UK) to remove CO₂ from theair entering the cuvette. Leaves were illuminated using a red-blue lightsource attached to the gas-exchange system, and light levels weremaintained at saturating photosynthetic photon flux density (PPFD) of1000 μmol m⁻² s⁻¹ with an integral LED light source (PP systems) for theduration of the A/Ci response curve. Measurements of A were made atambient CO₂ concentration (C_(a)) at 400 μmol mol⁻¹, before C_(a) wasdecreased to 300, 200, 150, 100, 50 μmol mol⁻¹ before returning to theinitial value and increased to 500, 600, 700, 800, 900, 1000, 1100, 1200μmol mol⁻¹. Leaf temperature and vapour pressure deficit (VPD) weremaintained at 22° C. and 1±0.2 kPa respectively. The maximum rates ofRubisco—(Vc_(max)) and the maximum rate of electron transport for RuBPregeneration (J_(max)) were determined and standardized to a leaftemperature of 25° C. based on equations from Bernacchi et al. (2001),and McMurtrie & Wang (1993), respectively.

PSI and PSII Quantum Efficiency

The photochemical quantum efficiency of PSII and PSI in transgenic andWT plants was measured following a dark-light induction transition usinga Dual-PAM-100 instrument (Walz, Effeltrich, Germany) with a DUAL-DRmeasuring head. Plants were dark adapted for 20 min before placing inthe instrument. Following a dark adapted measurement, plants wereilluminated with 220 μmol m⁻² s⁻¹ PPFD. The maximum quantum yield ofPSII was measured following a saturating pulse of light for 600 mssaturating pulse of light at an intensity of 6200 μmol m⁻² s⁻¹. The PSIIoperating efficiency was determined as described by the routines above.PSI quantum efficiency was measured as an absorption change of P700before and after a saturating pulse of 6200 μmol m⁻² s⁻¹ for 300 ms(which fully oxidizes P700) in the presence of far-red light with a FRpre-illumination of 10 s. Both measurements were recorded every minutefor 5 min). q_(p) or (F_(v)′/F_(m)′), was calculated from measurementsof steady state fluorescence in the light (F′) and maximum fluorescencein the light (F_(m)′) whilst minimal fluorescence in the light (F_(o)′)was calculated following the equation of Oxborough and Baker (1997b).The fraction of open PSII centres (q_(L)) was calculated fromq_(p)×F_(o)′/F (Baker 2008).

Pigment Extraction and HPLC Analysis

Chlorophylls and carotenoids were extracted using n,n-dimethylformamide(DMF) (Inskeep and Bloom 1985) which was subsequently shown tosuppressed chlorophyllide formation in Arabidopsis leaves (Hu et al.,2013). Briefly, leaf discs collected from two different leaves wereimmersed in DMF at 4° C. for 48 hours and separated by UPLC as describedby Zapata et al., (2000).

Determination of Sucrose and Starch

Carbohydrates and starch were extracted from 20 mg leaf tissue andsamples were collected at 2 time points, 1 hour before dawn (15 h intothe dark period) and 1 hour before sunset (7 h into the light period).Four leaf discs collected from two different leaves were ground inliquid nitrogen and 20 mg/FW of tissue was incubated in 80% (v/v)ethanol for 20 min at 80° C. and then repeated 3 times with ethanol 80%(v/v) at 80° C. The solid pellet and pooled ethanol samples and freezedried. Sugars were measured from the extracts in ethanol using anenzyme-based protocol (Stitt et al., 1989), and the starch contents wereestimated from the ethanol-insoluble pellet according to Stitt et al.(1978), with the exception that the samples were boiled for 1 h and notautoclaved.

Statistical Analysis

All statistical analyses were done by comparing ANOVA, using Sys-stat,University of Essex, UK. The differences between means were tested usingthe Post hoc Tukey test (SPSS, Chicago).

Results

Production and Selection of Arabidopsis Transformants

The full-length tobacco Rieske iron sulphur coding sequence of thecytochrome b6f complex (Cyt b6f: X64353) was used to generate anover-expression construct B2-NtRi (FIG. 9). Following floral dipping,Arabidopsis plants were regenerated on kanamycin/hygromycin containingmedium and plants expressing the integrated transgenes were screenedusing RT-PCR (data not shown).

Total extractable protein from leaves of the T1 progeny was analysed andthree lines identified showing a significant over-expression of theRieskeFeS protein (PetC) (FIG. 10A). Immunoblot analysis of T3 progenyfrom selected lines for Arabidopsis expressing RieskeFeS carried outusing WT as controls. Immunoblot analysis revealed that all three plantslines selected in the T1 generation showed an accumulation of theRieskeFeS protein (FIG. 1A). Interestingly, the over-expression ofRieskeFeS (referred to as Rieske FeS ox) resulted in a concomitantincrease in cyt f (PetA) and cyt b₆ (PetB), two proteins found withinthe cytochrome b6f complex (FIG. 1A).

An increase in the level of the PSI type I chlorophyll a/b-bindingprotein (Lhcal) and an increase in the core protein of PSI (PsaA) wasalso observed. Furthermore, the DI (PsbA) and D2 (PsbD) proteins whichform the reaction centre of PSII were shown to be elevated in Rieske FeSox lines. Finally, an increase in the ATP synthase delta subunit (AtpD)was also observed in Rieske FeS ox lines (FIG. 1A). As a control forprotein loading and expression level (FIG. 1A), the samples were probedwith antibodies to the plastidial Calvin-Benson Cycle enzyme FBPaldolase (FBPA) and the mitochondrial photo-respiration enzyme glycinedecarboxylase H-subunit (GDCH). No significant differences in proteinlevels for either FBPA or GDCH were observed. Furthermore, nosignificant differences in the levels of Rubisco were observed betweentransgenic and WT plants (FIG. 1A). A quantitative estimate of thechanges in protein levels was determined from the immunoblots of samplescollected from two to three independent plants per lines. An example isshown in FIG. 1B. These results showed a 2-2.5 fold increase in theRieske FeS protein relative to WT plants and a similar increase was alsoobserved for cyt f, cyt b₆, Lhca1, D2 and PsaA (FIG. 1C). No increase inthe stromal FBPA protein was evident.

Chlorophyll Fluorescence Imaging Reveals Increased PhotosyntheticEfficiency in Young Transgenic Seedlings

In order to screen for photosynthetic changes in seedlings (T3 progeny)grown at either 130 μmol m⁻² s⁻¹ chlorophyll a fluorescence imaging wasused to examine the quantum efficiency of PSII photochemistry(F_(q)′/F_(m)′) (Baker, 2008; Murchie and Lawson, 2013). Analysis ofplants over-expressing the cytochrome b6f complex grown at 130 μmol m⁻²s⁻¹, showed a small increase in F_(q)′/F_(m)′ at an irradiance of 310μmol m⁻² s⁻¹ (FIG. 2a ) and 450 μmol m⁻² s⁻¹ (FIG. 2b ) when compared toWT. At higher light level (600 μmol m⁻²s⁻¹), significant increase inF_(q)′/F_(m)′ were still observed (FIG. 11a ). Furthermore, nosignificant differences in Fv/Fm were observed between transgenic and WT(FIG. 11b ). From images taken at the time of fluorescence analysis ofthe seedlings it was shown that the leaf area for all transgenic lineswas significantly larger than WT (FIG. 2d ). Wild type plants used inthis study were a combined group of WT and null segregants verified byPCR. Interestingly, line 11, which showed the largest increase inF_(q)′/F_(m)′, also showed the biggest increase in leaf area in allexperiments. Furthermore, a more detailed analysis of photosyntheticparameters by fluorescence imaging demonstrated a consistent increase inthe operating efficiency of PSII (F_(q)′/F_(m)′; FIG. 15), a decrease innon-photochemical quenching (NPQ; FIG. 2 c; FIG. 16) and a significantincrease in the maximum potential operating efficiency of PSII in thelight (F_(v)′/F_(m)′; FIG. 17). Wild-type plants used in this study werea combined group of WT and null segregants from the rieskeFeSover-expressing lines verified by PCR. A comparison of wild type plantsand null-segregants clearly showed that no significant differences wereobserved in either photosynthetic efficiency (F_(q)′/F_(m)′) or in leafarea between the two groups (FIG. 18).

Evaluation of the Relationship Between Carbon Assimilation andFluorescence at 21% O₂

Light response curves conducted to assess the relationship between thephotosynthetic operating efficiency (F_(q)′/F_(m)′) and CO₂ fixationcorrected for leaf fractional light absorbance (ϕCO₂). This provides ameasure of the efficiency of light utilization for CO₂ fixation. FIG. 3shows the light response curves carried for each lines independently(FIG. 3a ) and all lines combined (FIG. 3b ) compared to wild type. Therelationship between the photosynthetic operating efficiency and yieldof CO₂ assimilation enables an assessment of possible alternativeelectron sinks to Rubisco activity.

Both the operating efficiency of PSII (F_(q)′/F_(m)′ ϕCO₂) (FIG. 3c )and the maximum quantum efficiency of CO₂ fixation (ϕCO₂) (FIG. 3d )were shown to be elevated compared to wild type when analysed at 21% O₂.However, these differences were only significant in line 11 and when alllines were combined into a single group (ALL). Additionally, nosignificant differences in average stomatal conductance were observed intransgenic lines compared to wild type (FIG. 3e ).

Evaluation of the Relationship Between Carbon Assimilation andFluorescence at 2% O₂

In addition to light response curves evaluated at 21% O₂,non-photorespiratory conditions (20 mmol mol⁻¹ O₂) were used for thelight response curves to further assess the relationship between thephotosynthetic operating efficiency (F_(q)′/F_(m)′) and CO₂ fixationcorrected for leaf fractional light absorbance (ϕCO₂). This provides ameasure of the efficiency of light utilization for CO₂ fixation.

FIG. 4 shows the light response curves carried out undernon-photorespiratory conditions for each lines independently (FIG. 4a )and all lines combined (FIG. 4c ) compared to wild type. The rate ofelectron transport (rETR) for each line independently compared to wildtype is also shown (FIG. 4b ). The rETR was shown to be increased incomparison to wild type. The relationship between the photosyntheticoperating efficiency and yield of CO₂ assimilation enables an assessmentof possible alternative electron sinks to Rubisco activity. Both theoperating efficiency of PSII (F_(q)′/F_(m)′ ϕCO₂) (FIG. 4d ) and themaximum quantum efficiency of CO₂ fixation (ϕCO₂) (FIG. 4e ) were shownto be significantly elevated compared to wild type. Additionally, onlyminor differences in stomatal conductance were observed in one of thetransgenic lines (11) compared to wild type (FIG. 4f ).

Increased Quantum Efficiency of PSI and PSII in Comparison to Wild Type

To further explore the influence of increases in the Rieske FeS proteinon PSII and PSI photochemistry dark-light induction responses weredetermined in WT and Rieske FeS ox transgenic plants using simultaneousmeasurements of P700 oxidation state and PSII efficiency.

FIG. 5 shows that the quantum efficiency of both PSI and PSII wereincreased in the Rieske FeS ox plants compared to wild type and that thefraction of PSII centres that were open (q_(L)) was also increasedwhilst the level of Qa reduction (1-qP) was lower in leaves of 27 dayold plants. NPQ level was also lower in the Rieske FeS ox plantsrelative to wild type (FIG. 5A). Furthermore, a reduction in stressinduced limitation of NPQ (q_(N)) was also observed.

Increased Cytochrome b6f Protein Levels Stimulates Growth in Low Light

The same group of plants used for fluorescence analysis described abovewere assembled and photographed (FIG. 7C). Their growth rate wasdetermined by evaluating leaf area over 26 days from planting until sucha time that aerial observation became complex due to overlapping leafareas obscuring evaluated differences. From image analysis of theseedlings it was shown that the leaf area for all lines wassignificantly greater than WT as early as 3 days after planting ontosoil (FIGS. 7A and D). By 16 days, plants over-expressing the Rieske FeSprotein were shown to be between 40-114% larger than corresponding wildtype plants. Furthermore, line 11 was shown to be significant largerthan lines 9 and 10. After 25 days post-planting the plants weredestructively harvested and dry weight were determined. Lines 9 to 11respectively showed a 29%, 46% and 72% increase in biomass compared toWT.

Photosynthetic CO₂ Assimilation Rates are Increased in Mature Plants.

The rate of CO₂ assimilation (A) was determined in plants grown at 130μmol m⁻²s⁻¹ as a function of internal CO₂ concentration (C_(i)) in youngexpanding leaves. Under these experimental growth conditions, aspreviously used for light response curves, no significant differences inCO₂ assimilation, J_(max) or Vc_(max) were observed (data not shown). Asecond group of plants growing at 280 μmol m⁻²s⁻¹ in the green housemaintained in square wave light under isolights with a 12 h/12 h daynight cycle were also examined (FIG. 6). Under these higher lightconditions, in all transgenic plants the rate of A in developing leaveswas greater at C_(i) concentrations above ca. 300 μmol mol⁻¹ whencompared to WT plants (FIG. 6A-B) resulting in a greater light saturatedrate of photosynthesis (A_(sat)) in lines 9 and 11 compared with the WTcontrol (FIG. 6C). A_(sat) was clearly elevated in lines ALL compared toWT. Further analysis of the A/C_(i) curves illustrated that significantenhancements of the maximum rate of Rubisco carboxylation (Vc_(max):FIG. 6D) and electron transport (J_(max): FIG. 6E) were evident in somelines and significantly elevated in ALL. Further analysis of the A/C_(i)curves illustrated that the light- and CO₂-saturated rate ofphotosynthesis (A_(max)) was also significantly greater in 2 of the 3transgenic lines (FIG. 6C). An analysis of final leaf area and final dryeight (biomass) of the plants used here showed an overall significantincrease in both leaf area (FIG. 7A) and final biomass (FIG. 7B)

Increased cytochrome b6f protein levels stimulates growth in low light.The same group of plants used for fluorescence analysis described abovewere assembled and photographed (FIG. 8A). Their growth rate wasdetermined by evaluating rosette area over 26 days from planting untilsuch a time that aerial observation became complex due to overlappingleaf areas obscuring evaluated differences. From image analysis of theseedlings it was shown that the leaf area for all lines wassignificantly greater than WT as early as 8 days after planting ontosoil (FIG. 8B). By 16 days, plants over-expressing the RieskeFeS proteinwere shown to be between 40-114% larger than corresponding wild typeplants. Furthermore, line 11 was shown to be significant larger thanlines 9 and 10.

After 25 days post-planting the plants were destructively harvested anddry weight were determined. Lines 9 to 11 respectively showed a 29%, 46%and 72% increase in biomass compared to WT (FIG. 8C).

Over-Expression of the RieskeFeS Protein Results in Changes to thePigment Content of Transgenic Lines.

Four leaf discs from two different leaves from selected lines werecollected and the pigments were extracted using DMF and pigments wereseparated by UPLC as described by Zapata et al., (2000). An average 26%increase in chlorophyll content was observed in transgenic lines. Theseincreases were accompanied by an increase in neoxanthin (+38%),violaxanthin (+59%), lutein (+75%) and β-carotene (+169%). Chlorophyllratios of approx 3.05-3.10 in both WT and transgenic lines is similar tothe 3.11 previously reported in coffee leaves and 2.94 in green coffeecherries (Simkin et al., 2008; 2010). Interestingly, line 11 (previouslyshown to have an overall higher increase in PSII efficiency (FIG. 2),higher average increases in A_(max) (FIGS. 3, 4 and 6) and a fastergrowth rate (FIG. 8) was shown to have the highest increase in bothchlorophyll and carotenoids compared to WT (see Table 1).

TABLE 1 shows pigment content in WT and transgenic lines. Results arerepresented as units of β-carotene in WT (where β-carotene in WT = 1).WT 9 10 11 ALL Neoxanthin 0.90 +/− 0.08  1.22 +/− 0.08*  1.17 +/− 0.08* 1.33 +/− 0.10**  1.24 +/− 0.05** Violaxanthin 0.92 +/− 0.08  1.42 +/−0.08***  1.39 +/− 0.10***  1.58 +/− 0.10***  1.46 +/− 0.05***Violaxanthin  +54%  +51%  +71%  +55% Lutein 3.09 +/− 0.25  5.36 +/−0.40***  5.05 +/− 0.31***  5.84 +/− 0.28***  5.42 +/− 0.20*** Lutein +73%  +63%  +89%  +75% β-carotene 1.00 +/− 0.10  2.71 +/− 0.19***  2.45+/− 0.18***  2.92 +/− 0.08***  2.69 +/− 0.10*** β-carotene +171% +145%+192% +169% total car 5.91 +/− 0.44 10.06 +/− 0.64*** 10.71 +/− 0.55***11.67 +/− 0.42*** 10.81 +/− 0.35*** chl/a 15.56 +/− 0.50  19.82 +/−1.01*** 18.31 +/− 0.95 20.83 +/− 0.79*** 19.65 +/− 0.56*** chl/b 5.11+/− 0.17  6.41 +/− 0.27**  5.87 +/− 0.27*  6.73 +/− 0.32***  6.34 +/−0.18*** chl total 20.67 +/− 0.61  26.23 +/− 1.26*** 24.18 +/− 1.21**27.55 +/− 1.10*** 27.55 +/− 1.10*** chlorophyll  +27%  +17%  +33%  +26%ratio chl a/b 3.05 3.09 3.12 3.10 3.10 chl/βC 20.67  9.68 9.87 9.43 9.65chl/Lutien 6.68 4.89 4.79 4.72 4.8 Statistical differences are shown inbold (*<0.1; **<0.05; ***<0.001).

Discussion

The primary determinant of plant productivity is associated directlywith photosynthetic efficiency and any improvements, through geneticmanipulation or otherwise, can greatly influence biomass and yield.

Certain embodiments of the present invention illustrate that increasingthe level of the RieskeFeS protein component of the cyt 6bf complexresults in a co-committant increase in other components of the complex,PetA and PetB. Certain embodiments of the present invention may alsoresult in a co-committant increase in the PSI type I chlorophylla/b-binding protein (Lhcal) and the core protein of PSI (PsaA) and PsbA,PsbD of the PSII reaction centre and the ATP synthase delta subunit(AtpD). The combined increase of the cyt b6f complex and otheridentified proteins results in a substantial and significant impact onphotosynthesis and biomass of Arabidopsis grown under standard growthroom conditions.

Chlorophyll fluorescence imaging used to analyse plants at 14 dayspost-planting demonstrated that the positive effect of the manipulationon the cyt b6f complex is evident at an early stage of development.Interestingly, lines 11, which showed the largest overall increases inphotosynthetic efficiency (F_(q)′/F_(m)′ and A_(max)) also showed thelargest increase in leaf area and biomass when compared to WT.

CONCLUSION

As demonstrated herein, over-expression of RieskeFeS protein, a keycomponent of the cytobrome b6f complex, resulted in a co-commitantincrease in the protein levels of several other components of thecomplex as well as other proteins involved electron transport and plantgrowth.

Throughout the description and claims of this specification, the words“comprise” and “contain” and variations of them mean “including but notlimited to” and they are not intended to (and do not) exclude othermoieties, additives, components, integers or steps. Throughout thedescription and claims of this specification, the singular encompassesthe plural unless the context otherwise requires. In particular, wherethe indefinite article is used, the specification is to be understood ascontemplating plurality as well as singularity, unless the contextrequires otherwise.

Features, integers, characteristics or groups described in conjunctionwith a particular aspect, embodiment or example of the invention are tobe understood to be applicable to any other aspect, embodiment orexample described herein unless incompatible therewith. All of thefeatures disclosed in this specification (including any accompanyingclaims, abstract and drawings), and/or all of the steps of any method orprocess so disclosed, may be combined in any combination, exceptcombinations where at least some of the features and/or steps aremutually exclusive. The invention is not restricted to any details ofany foregoing embodiments. The invention extends to any novel one, ornovel combination, of the features disclosed in this specification(including any accompanying claims, abstract and drawings), or to anynovel one, or any novel combination, of the steps of any method orprocess so disclosed.

The reader's attention is directed to all papers and documents which arefiled concurrently with or previous to this specification in connectionwith this application and which are open to public inspection with thisspecification, and the contents of all such papers and documents areincorporated herein by reference.

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1. A method of increasing a photosynthesis rate of a plant, the methodcomprising expressing within said plant an exogenous Rieske iron sulphurprotein to form a transgenic plant.
 2. (canceled)
 3. A method accordingto claim 1, wherein: (i) the transgenic plant has an increasedphotosynthesis rate as compared to a control plant; (ii) the transgenicplant has a greater size, greater biomass and/or faster growth rate ascompared to a control plant or (iii) the transgenic plant has anenhanced yield as compared to a control plant: wherein the control plantis optionally a wild-type plant of the same variety as the transgenicplant. 4-9. (canceled)
 10. A method according to claim 1, wherein themethod further comprises: (i) a step of nuclear transformation of aplant cell with a nucleic acid molecule encoding the exogenous Rieskeiron sulphur protein; (ii) introducing into at least one plant cell anexpression vector comprising a nucleic acid molecule encoding theexogenous Rieske iron sulphur protein; and optionally regenerating atransgenic plant from said at least one plant cell; (iii) the step ofcultivating the transgenic plant under conditions suitable forphotosynthesis to occur; and/or (iv) the step of determining anincreased photosynthesis rate of the transgenic plant. 11-14. (canceled)15. A method according to claim 1, which further comprises cultivatingthe transgenic plant under conditions promoting plant growth anddevelopment; optionally wherein: (i) a progeny of said transgenic plantis obtained; (ii) the method further comprises the step of cultivatingthe progeny of the transgenic plant under conditions suitable forphotosynthesis to occur; (iii) the method further comprises the step ofdetermining an increased photosynthesis rate of the progeny of thetransgenic plant; (iv) the method further comprises the step ofobtaining a progeny of said transgenic plant comprises selfing thetransgenic plant thereby producing a plurality of first generationprogeny plants; and/or (v) the method further comprises the step ofcultivating the progeny of the transgenic plant under conditionssuitable for photosynthesis to occur, optionally, which furthercomprises the step of determining an increased photosynthesis rate ofthe plurality of first generation progeny plants. 16-20. (canceled) 21.The method according to claim 1, the method comprising increasingexpression of a cytochrome b6f complex protein in at least one plantcell, the method comprising: introducing into the at least one plantcell a nucleic acid molecule encoding an exogenous Rieske iron sulphurprotein; and expressing said Rieske iron sulphur protein in said plantcell.
 22. A method according to claim 21, wherein: (i) the cytochromeb6f complex protein is selected from PetA, PetB and a combination ofPetA and PetB; (ii) the cytochrome b6f complex protein is an endogenouscytochrome b6f complex protein; (iii) the step of introducing into atleast one plant cell a nucleic acid molecule encoding an exogenousRieske iron sulphur protein comprises introducing an expression vectorcomprising the nucleic acid molecule encoding the exogenous Rieske ironsulphur protein; (iv) which further comprises the step of the step ofcultivating the transgenic plant under conditions suitable forphotosynthesis to occur; and/or (v) which further comprises the step ofdetermining an increased photosynthesis rate of the transgenic plant.23-26. (canceled)
 27. A method according to claim 21, which furthercomprises regenerating a transgenic plant from said at least one plantcell comprising said nucleic acid molecule, optionally, which furthercomprises: (i) selecting a transgenic plant having an increasedexpression of a cytochrome b6f complex protein relative to acorresponding control plant; (ii) selecting a transgenic plant having anincreased photosynthesis rate relative to a corresponding control plant;and/or (iii) a method wherein the control plant is a wild-type plant ofthe same variety as the transgenic plant. 28-30. (canceled)
 31. A methodaccording claim 1, wherein the exogenous Rieske iron sulphur proteincomprises an amino acid sequence selected from: a) an amino acidsequence as set forth in SEQ. ID. No.
 1. 32. A method according to claim1, wherein the exogenous Rieske iron sulphur protein is a protein havingan amino acid sequence which has at least 70% sequence identity to theamino acid set forth in SEQ. ID. No.
 1. 33. A method according to claim10, which comprises introducing into the at least one cell a nucleicacid molecule comprising a nucleic acid sequence selected from: a) anucleic acid sequence as set forth in SEQ. ID. No. 2 or SEQ. ID. No. 3;b) a nucleic acid sequence having at least 70% sequence identity to thenucleic acid sequence as set forth in SEQ. ID. No. 2 or SEQ. ID. No. 3and which encodes a Rieske iron sulphur protein; c) a nucleic acidsequence which hybridises to (a) or (b) and which encodes a Rieske ironsulphur protein; and d) a nucleic acid sequence which differs from (a),(b) or (c) by virtue of the degeneracy of the genetic code and whichencodes a Rieske iron sulphur protein.
 34. A method according to claim1, wherein the nucleic acid molecule is an isolated nucleic acidmolecule.
 35. A method according to claim 11, wherein the expressionvector is a plasmid suitable for nuclear transformation.
 36. A chimericconstruct for increased expression of a cytochrome b6f protein in aplant, the chimeric construct comprising a nucleic acid molecule, whichencodes a Rieske iron sulphur protein, operably linked to a promoterwhich is operable in a plant cell, wherein the nucleic acid moleculecomprises a nucleic acid sequence selected from: i) a nucleic acidsequence as set forth in SEQ. ID. No. 2 or SEQ. ID. No. 3; ii) a nucleicacid sequence having at least 70% sequence identity to the nucleic acidsequence as set forth in SEQ. ID. No. 2 or SEQ. ID. No. 3 and whichencodes a Rieske iron sulphur protein; iii) a nucleic acid sequencewhich hybridises to (a) or (b) and which encodes a Rieske iron sulphurprotein; and iv) a nucleic acid sequence which differs from (a), (b) or(c) by virtue of the degeneracy of the genetic code and which encodes aRieske iron sulphur protein.
 37. A plant cell comprising a nucleic acidmolecule comprising a nucleic acid sequence selected from: a) a nucleicacid sequence as set forth in SEQ. ID. No. 2 or SEQ. ID. No. 3; b) anucleic acid sequence having at least 70% sequence identity to thenucleic acid sequence as set forth in SEQ. ID. No. 2 and which encodes aRieske iron sulphur protein; c) a nucleic acid sequence which hybridisesto (a) or (b) and which encodes a Rieske iron sulphur protein; and d) anucleic acid sequence which differs from (a), (b) or (c) by virtue ofthe degeneracy of the genetic code, wherein the nucleic acid moleculeencodes an exogenous Rieske iron sulphur protein, optionally wherein:(i) the nucleic acid molecule is comprised in an expression vector; (ii)the expression vector is suitable for nuclear transformation of thenucleic acid molecule; or (iii) the expression vector further comprisesa promoter such as a 35s tobacco mosaic virus promoter such as a 35stobacco mosaic virus promoter. 38-48. (canceled)
 49. A method ofincreasing a photosynthesis rate of a plant such as wheat, comprisingincreasing a level of expression of an endogenous cytochrome b6f complexprotein, wherein the endogenous cytochrome b6f complex protein comprisesRieske iron sulphur protein, optionally wherein the Rieske iron sulphurprotein: a) has an amino acid sequence having at least 70% sequenceidentity to the amino acid set forth in SEQ. ID. No. 1; b) is encoded bya nucleic acid sequence having at 5 least 70% identify sequence identityto the nucleic acid sequence as set forth in SEQ ID. No. 2 or SEQ ID No.3; c) is encoded by a nucleic acid sequence which hybridises to (a) or(b); or d) is encoded by a nucleic acid sequence which differs from (b)or (c) by virtue of the degeneracy of the genetic code.
 50. A plant orplant cell obtainable by the method of claim
 49. 51. The method of claim49, wherein the plant is wheat.
 52. A vector for upregulating one ormore components of a cytochrome b6f complex in a plant, wherein the oneor more components of the cytochrome b6f complex comprises Rieske ironsulphur protein.
 53. The vector according to claim 51, wherein thevector is adapted for expression in a plant cell and/or a plasmid.